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Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O2 activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O2 with the mixed valence form (CuA(2+), heme a(3+), heme a3(2+)-CuB(1+)) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm(-1) at the peroxy oxidation level (PM). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the PM intermediate is also formed in the reaction of Y167F with O2. These results suggest that in the fully reduced enzyme, the proton pumping ν(Fe(IV)=O) = 804 cm(-1) to ν(Fe(IV)=O) = 790 cm(-1) transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a3 but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a3. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a3-Asp-399-H2O site and, thus, to the exit/output proton channel (H2O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a3 controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.
Background: Understanding the coupling of O2 reduction to proton pumping by CcO requires detection of reaction intermediates.
Results: We have detected two oxoferryl intermediates at the PM oxidation state.
Conclusion: The H-bonding properties of the proximal heme a3 His ligand control the strength of the oxoferryl species.
Significance: The role of His-411, Thr-389, Gly-386, and Asp-399 residues in the proton pumping P→F transition is outlined.
A low potential electron carrier ferredoxin (E0′ ≈ −500 mV) is used to fuel the only bioenergetic coupling site, a sodium-motive ferredoxin:NAD+ oxidoreductase (Rnf) in the acetogenic bacterium Acetobacterium woodii. Because ferredoxin reduction with physiological electron donors is highly endergonic, it must be coupled to an exergonic reaction. One candidate is NADH-dependent caffeyl-CoA reduction. We have purified a complex from A. woodii that contains a caffeyl-CoA reductase and an electron transfer flavoprotein. The enzyme contains three subunits encoded by the carCDE genes and is predicted to have, in addition to FAD, two [4Fe-4S] clusters as cofactor, which is consistent with the experimental determination of 4 mol of FAD, 9 mol of iron, and 9 mol of acid-labile sulfur. The enzyme complex catalyzed caffeyl-CoA-dependent oxidation of reduced methyl viologen. With NADH as donor, it catalyzed caffeyl-CoA reduction, but this reaction was highly stimulated by the addition of ferredoxin. Spectroscopic analyses revealed that ferredoxin and caffeyl-CoA were reduced simultaneously, and a stoichiometry of 1.3:1 was determined. Apparently, the caffeyl-CoA reductase-Etf complex of A. woodii uses the novel mechanism of flavin-dependent electron bifurcation to drive the endergonic ferredoxin reduction with NADH as reductant by coupling it to the exergonic NADH-dependent reduction of caffeyl-CoA.